Emerging evidence indicates that many non-histone proteins, such as p53, DNMT1, E2F1, NF B, RB, and STAT3, are methylated at specific lysine residues to control their protein stability or activity. A major outcome of these site-specific methylations is to trigger the destruction of modified proteins through the ubiquitin-proteasome system. However, the identity of the ubiquitin E3 ligase and the mechanism it employs to specifically recognize and target the methylated protein substrates for proteolysis remain unclear. We propose to investigate how the methylated DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is targeted for degradation by a novel CRL4 ubiquitin E3 ligase to establish the mechanism by which methylated non-histone proteins are destroyed in a rapid and temporally regulated fashion. DNMT1 is a major DNA methyltransferase that maintains the CpG methylation patterns during DNA replication. The DNMT1-mediated methylation on the hemi-methylated cytosine residues in newly synthesized DNA strand is a main mechanism to preserve epigenetic inheritance that defines cell- or tissue-specific gene expression. DNMT1 is methylated on specific lysines which are dynamically regulated by SET7 methyltransferase and LSD1 demethylase. Loss of LSD1 increases the levels of the methylated DNMT1 protein and triggers the protein destruction of DNMT1. Our studies found that the methylation-dependent destruction of DNMT1 protein is controlled by a novel CRL4 ubiquitin E3 ligase. We propose to establish the mechanism by which CRL4 recognizes the methylated form of DNMT1 and promotes the modified DNMT1 for degradation with following specific aims: 1) To establish the involvement of CRL4 in mediating the methylation-specific destruction of DNMT1; 2) To identify the substrate-receptor subunit(s) of CRL4 that specifically recognizes the methylated form of DNMT1 and directly interacts with the domain that contains the methyl group; 3) To examine how DNMT1 degradation is regulated in the cell cycle and coupled to DNA replication to faithfully maintain epigenetic inheritance. We believe our substantial research experiences on CLR4 ubiquitin E3 ligases and LSD1- mediated histone methylation should provide strong background on achieving the proposed goals for this important research. As increasing number of non-histone proteins are found to be methylated, our studies should reveal novel insights into the mechanism by which protein methylation is coupled to the regulation of cell growth and development, and how the dysfunction of this modification can cause various human diseases. In addition, as this is an R15 grant application, we hope to mentor graduate and undergraduate students on how to conduct a high impact research.